Plant Cell Reports (1996) 15:691-694 Plant Cell Reports 9 Springer-Verlag 1996 Forskolin synthesis in in vitro cultures of Coleus forskohlii Briq transformed with Agrobacterium tumefaciens Swapna Mukherjee, Biswajit Ghosh, and Sumita Jha Centre of Advanced Study, Dept. Botany, Calcutta University, 35, Ballygunge Circular Road, Calcutta 700019, India Received 30 March 1995/Revised version received 8 November 1995 - Communicated by F. Constabel Abstract. Agrobacterium tumefaciens mediated tumor tissue and shooty teratomas of Coleus forskohlii were cultured in vitro. Forskolin was detected in tumorous callus (0.002%), rhizogenic callus (0.011%) and root cultures (0.014 %), but not in shooty teratomas. Forskolin synthesis and accumulation in tumorous C. forskohlii cultures may permit the elucidation of diterpene metabolism in this species. Abbreviations. B 5, medium after Gamborg et al. (1968); 2,4-D, 2,4-dichlorophenoxyacetic acid; MS, medium after Murashige and Skoog (1962); BAP, benzylaminopurine; IBA, indole-3-butyric acid; Kn, kinetin; CH, casein hydrolysate; T-DNA, transferred DNA. Introduction Crown galls and gall derived cell suspension cultures have been found to synthesize secondary metabolites such as quinoline alkaloids in Cinchona ledgeriana (Payne et al. 1987), polyacetylenes in Bidens sp. (Norton and Towers 1985; Towers 1989), isoflavonoid glucosides in Lupinus sp. (Berlin et al. 1989), and cardenolides in Digitalis lanata (Moldenhauer et al. 1990). Shooty teratomas induced with turnout-inducing (Ti) plasmids in Mentha species have been reported to produce monoterpenes (Spencer et al. 1990a, 1990b). In Nicotiana tabacum, shooty teratomas have shown the ability to biotransform nicotine and nornicotine (Saito et al. 1989). Biotransformation of hyoscyamine to scopolamine by shooty teratomas of Atropa belladonna have also been reported (Saito et al. 1991) Coleus forskohlii Briq. (Lamiaceae) is an important plant in Indian Ayurvedic medicine. It produces the labdane diterpenoid forskolin in its tuberous roots (Bhat et al. 1977) Forskolin effects heart action (positive inotropic effect), lowers blood and intraocular pressure and has anti-inflammatory properties. The sole source of forskolin is still the roots of wild or cultivated C. forskohlii plants. There are some reports on in vitro culture of C. forskohlii tissues for production of forskolin (Mersinger et al. 1988; Krombholz et al. 1992; Sen et al. 1992). Mersinger et al. (1988) and Krombholz et al. (1992) reported a close correlation between root differentiation and forskolin production. Sen et al. (1992) on the other hand, demonstrated the production of forskolin in shoots and shoot forming callus, but not or only traces in rhizogenic caltus and root cultures, thus showing that root differentiation and forskolin production are not necessarily coupled. We were interested in determining the potential of tumorous cultures for production of forskolin in C. forskohlii. Here we report initiation of transformed cultures of C. forskohlii and provide new evidence of root tissue as the site of forskolin synthesis and accumulation. Material and Methods Plant Material. Plants of Coleusforskohlii Briq. were obtainedfrom the National Bureau of Plant Genetic Resources, New Delhi, India and grown in the Experimental Garden, Department of Botany, University of Calcutta. Production of Plantlets as Source of Explants. Shoottips (1.0-1.5 cm) were washed with 5% Teepol detergent for 10 min, disinfected with 0.1% mercuric chloride for 10 rain and rinsed with sterile distilled water. The shoot tips were then cultured on solidified Murashige and Skoog's (1962) medium with 3% sucrose and without hormones. The pH of the media had been adjusted to 5.6 prior to autoclaving at 121~ 1.05 kg/cm 2, for 15 rain. Cultures were kept under 1618h light/dark cycles of cool white fluorescent 40W tubes providing 37.5 /~molm-2s l light intensity and at 25~ + 1~ The shoot tips proliferated, were multiplied and subcultured on MS mediumcontaining Kn (1 mg la). Individual shoots were excised and rooted in MS medium without growth regulators (MSO) within 2-3 weeks. When 8- 10 weeks old, resulting plantlets were used for A. tumefaciens mediated tumourproductionand subsequentforskolin analysisof tumoroustissues. Correspondence to: S. Jha